Classifications

• • C—CHEMISTRY; METALLURGY
  • C40—COMBINATORIAL TECHNOLOGY
  • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
  • C40B30/00—Methods of screening libraries
  • C40B30/04—Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins

Definitions

• This invention pertains to methods of screening a mixture for active entities.
• ICAT involves the site-specific, covalent labeling of proteins with isotopically normal or heavy reagents to quantitate levels of protein expression.
• Yet another example of methodology that separates and identifies proteins is a global version of the yeast two-hybrid screening assay developed by Uetz et al. (Uetz et al., Nature, 403(6770): 623-627 (2000)) and Ito et al. (Ito et al., Proc. Natl. Acad. Sci. USA, 98(8): 4569-4574 (2001)), which identified over 4,000 protein- protein interactions in Saccharomyces cerevisiae. Although these approaches for separating and identifying proteins are powerful, they do not identify the cellular functions of the fractionated proteins.
• Ligands to multiple entities can be detected using beads immobilized on an adhesive in combination with a subtractive screening method. This is referred to as the QuASAR method (International (PCT) Patent Application WO 01/40265) and was used to detect ligands that bound to virus and prion protein.
• the entity protein in its normal physiological environment is incubated with a peptide ligand library bound to beads.
• the beads are immobilized and arrayed in a thin gel of low melting-point agarose to create a crude "array" of ligands.
• a protein-binding membrane (nitrocellulose or PVDF) is laid on the gel, and the proteins are eluted from the beads under a variety of conditions and captured in the same relative position as they were in the gel on the membrane by unidirectional capillary transfer of solvent diffusing through the gel (similar to a Southern transfer of DNA).
• the membranes themselves can be stripped and reprobed for different, known proteins.
• the method comprises (i) providing a plurality of ligands, wherein each ligand is attached to a support to form a plurality of ligand-support complexes, (ii) contacting the ligand-support complexes with a mixture comprising a plurality of entities under conditions that allow at least one entity to bind to at least one ligand-support complex, thereby forming at least one entity-ligand-support complex, (iii) separating at least one entity-ligand-support complex from the unbound entities, (iv) assaying at least one entity of at least one separated entity-ligand-support complex for an activity, (v) detecting the activity, and (vi) selecting at least one entity-ligand-support-complex having the entity, which exhibited the detected activity.
• the method comprises (i) providing a plurality of ligands, wherein each ligand is attached to a support to form a plurality of ligand-support complexes, (ii) contacting the ligand-support complexes with a mixture comprising a plurality of entities under conditions that allow at least one entity to bind to at least one ligand-support complex, thereby forming at least one entity-ligand-support complex, (iii) separating at least one entity-ligand-support complex from the unbound entities, (iv) separating at least one entity-ligand-support complex and the ligand-support complexes into pools, (v) dissociating at least one entity from at least one separated entity-ligand-support complex, (vi) removing from the pools the ligand-support complexes or the at least one dissociated entity of step (v), (vii) assaying at least one dissociated entity of step (v) for an activity, (viii) detecting the activity
• the method comprises (i) providing a plurality of ligands, wherein each ligand is attached to a support to form a plurality of ligand-support complexes, (ii) contacting the ligand-support complexes with a mixture comprising a plurality of entities under conditions that allow at least one entity to bind to at least one ligand-support complex, thereby forming at least one entity-ligand-support complex, (iii) separating at least one entity-ligand-support complex from the unbound entities, (iv) separating at least one entity-ligand-support complex and the ligand-support complexes into pools, (v) adding a semi-solid or viscous material to each pool, wherein the entity of at least one separated entity-ligand-support complex dissociates from the complex and diffuses into the material, thereby forming a concentration gradient of the entity, wherein the concentration of the entity gradually decreases as the distance from the ligand-support complex from which at entity
• the invention provides methods of screening a mixture for active entities.
• the method comprises (i) providing a plurality of ligands, wherein each ligand is attached to a support to form a plurality of ligand-support complexes, (ii) contacting the ligand-support complexes with a mixture comprising a plurality of entities under conditions that allow at least one entity to bind to at least one ligand-support complex, thereby forming at least one entity-ligand-support complex, (iii) separating the at least one entity-ligandsupport complex from the unbound entities, (iv) assaying the at least one entity of the at least one entity-ligand-support complex for an activity, (v) detecting the activity, and (vi) selecting the at least one entity-ligand-support-complex having the entity, which exhibited the detected activity.
• mixture refers to any collection comprising more than one entity, wherein the term “entity” as used herein refers to any biological, chemical, or biochemical entity or target, such as a compound, molecule, virus, or cell.
• entity refers to any biological, chemical, or biochemical entity or target, such as a compound, molecule, virus, or cell.
• the mixture is a collection of different entities, each having a different chemical identity, e.g., molecular formula, chemical structure, nucleotide sequence or amino acid sequence, or each having a different physical identity, e.g., spectral signal or conformation. More preferably, the mixture is a collection comprising a plurality of different entities.
• the mixture can comprise one or more copies of each entity having a different chemical or physical identity.
• the entities comprising the mixture can be isolated from nature or synthetically produced, and can be organic or inorganic in nature (e.g., a synthetic inorganic compound or a synthetic organic compound).
• the entity can be a drug or drug candidate (such as a small molecule drug candidate), a fertilizer component, an insecticide component, or a derivative, analogue, or enantiomer thereof.
• the entity can be endogenous or exogenous to any prokaryote or eukaryote, e.g., a bacterium, a fungus, yeast, a plant, or a mammal.
• Suitable entities for the inventive method include, but are not limited to, cells (e.g., stem cells or cells in culture), proteins, peptides, drugs, antibodies, synthetic molecules, organic compounds, protein complexes (e.g., blood clotting Factor Xi ⁇ and fibrinogen or blood clotting Factor VIU and Von Willebrand Factor), bacteria, viruses, fungi, yeast, prions, amino acids, nucleic acids, carbohydrates, lipids, isoforms of any of the foregoing, and combinations of any of the foregoing.
• the entities are proteins.
• Suitable protein entities include, for example, receptors, antibodies, immunogens, enzymes (e.g., proteases), and enzyme substrates. More preferably, the proteins are plasma-derived proteins.
• the plasma-derived proteins are immunoglobulins, e.g., IgG, IgM, IgA, IgE.
• the immunoglobulins can be from an organism in a diseased state, (and optionally not found in the plasma of a healthy subject) or produced as a result of the administration of an agent, e.g., a drug.
• the entities are cells. More preferably, the cells are stem cells.
• the entity of the inventive methods can be obtained from any source, i.e., the mixture comprising the entities can be any complex mixture, such as extracts of soil, air, water, food, swabs for evaluating environmental contamination, intermediate or end-stage chemical reaction mixtures, and the like.
• the mixture comprising the entity can be a chemical or synthetic mixture and can be present in a combinatorial library and/or present in organic solvents under extreme conditions of pressure, temperature, etc.
• the mixture is a biological fluid, an environmental extract, or a composition comprising chemical compounds.
• biological fluid any aqueous solution that is derived from a prokaryotic or eukaryotic organism.
• the biological fluid can be obtained directly from the prokaryotic or eukaryotic organism, such as blood, lymph, tears, saliva, perspiration, and urine.
• the biological fluid can be obtained by culturing cells of the organism, such as fermentation broth and cell culture medium.
• Suitable biological fluids for use in the inventive method include, but are not limited to, blood, plasma, pooled plasma, intermediates of plasma fractionation, serum, a cell homogenate, a tissue homogenate, a conditioned medium, a fermentation broth, cerebrospinal fluid, urine, saliva, milk, ductal fluid, tears, perspiration, lymph, semen, umbilical cord fluid, and amniotic fluid.
• the biological fluid is a plasma-derived fraction comprising antibodies and anti- idiotype antibodies.
• anti-idiotype refers to an antibody that has an epitope that is specific for the antigen-determining region of another antibody.
• the biological fluid is obtained from a host afflicted with a disease.
• the term "host” as used herein refers to any eukaryotic or prokaryotic organism, e.g., bacteria, virus, yeast, fungi, bird, reptile, and mammal.
• the disease which afflicts the host, can be any disease including any condition, malady, infection, and the like.
• the disease can be cancer, diabetes, an autoimmune disease, osteoporosis, wound healing, liver regeneration, or lung disease.
• the disease can be an infection with a parasite, virus, or bacteria.
• the term "environmental extract” as used herein refers to any sample taken from an environment.
• the environment can be a natural environment, such as a naturally- occurring body of water.
• the environment can be a man-made environment, such as a building.
• the environmental extract can be a soil, soil extract, an extract from a naturally-occurring body of water, a sample of ice, air, ash, rock, or permafrost, or a swab from a building.
• the naturally-occurring body of water can be, for example, an ocean, a lake, a sea, a river, a swamp, a pond, a delta, or a bay.
• the environmental extract can alternatively be an extract from a water treatment center.
• the building can be any man-made building.
• the building is contaminated with one or more toxic agents, such as sarin, soman, nerve poisons, explosive chemicals, pesticides, pathogens, NX, and blister agents.
• composition comprising chemical compounds can comprise natural or synthetic chemical compounds.
• the composition can be a chemical or synthetic mixture of reaction products.
• the composition can be present in a combinatorial library and/or present in organic solvents under extreme conditions of pressure, temperature, etc.
• ligand refers to any biological, chemical, or biochemical agent, such as a compound, molecule, or cell that binds to an entity.
• the ligand can be isolated from natural or synthetically produced materials.
• the ligand can be endogenous or exogenous to a prokaryote or eukaryote, e.g. bacteria, a fungus, yeast, plant, or a mammal.
• Suitable ligands for the inventive methods include, but are not limited to, cells, bacteria, viruses, yeast, proteins, peptides, amino acids, nucleic acids, carbohydrates, lipids, drugs, synthetic inorganic compounds, synthetic organic compounds, antibody preparations (e.g., antibody fragments, chemically-modified antibodies, and the like), sugars, isoforms of any of the foregoing, and combinations of any of the foregoing.
• Organic molecules include, for example, synthetic organic compounds typically employed as pharmacotherapeutic agents. Such molecules are, optionally, mass-produced by combinatorial synthetic methods or, more specifically, by strategic syntheses devised to arrive at specific molecules.
• organic molecules also include natural products and analogues, whether extracted from their natural environment or strategically synthesized.
• the term "organic” as used herein is not intended to be limited to molecules comprised only of carbon and hydrogen, but rather is used in its broader sense as encompassing macromolecules of biological origin.
• the ligands are peptides.
• the term "peptide” as used herein refers to an entity comprising at least one peptide bond, and can comprise either D and/or L amino acids.
• the ligand is a peptide consisting essentially of about 2 to about 10 amino acids (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids).
• the peptide ligands are generated by combinatorial approaches, i.e., techniques commonly employed in the generation of a combinatorial library, e.g., the split, couple, recombine method or other approaches known in the art (see, e.g., Furka et al., Int. J.
• the random incorporation of 19 amino acids into pentapeptides produces up to 2,476,099 (19 5 ) individual peptides of differing sequence (Lam et al., supra).
• Combinatorial methods allow generation of libraries of ligands directly on a support.
• the ligands are synthesized on particles of support media such that multiple copies of a single ligand are synthesized on each particle (e.g., bead), although this is not required in the context of the invention.
• each of the ligands is attached to a support, thereby achieving formation of ligand-support complexes.
• support refers to any support matrix, such as those solid supports known in the art, which serve to immobilize the ligand.
• Suitable supports include, but are not limited to, membranes, filters, meshes, or particles comprising cellulose, acrylics, polyacrylamide or polyhydroxylated mefhacrylate polymers, polystyrene, dextran, agarose, polysaccharides, hydrophilic vinyl polymers, polymerized derivatives of any of the foregoing, and combinations of any of the foregoing, as well as any porous or non-porous matrix to which ligands can be directly attached or on which ligands can be synthesized.
• the support is inert such that chemical reaction with the entity and/or the immobilized ligand is minimized.
• the support desirably comprises a polymethacrylate, polyacrylate, agarose, a polyacrylamide, dextran, cellulose, a polysaccharide, nitrocellulose, silicon, styrene, metal, polyethylene-coated polystyrene, polyvinyldifluoride, nylon, or a combination of any of the foregoing.
• Particularly preferred support materials are polyethylene coated polystyrene and polymethacrylate.
• Various resins are commercially available, and, preferably, the support is a resin bead, such as a chromatographic resin bead.
• the ligands of the inventive method can be indirectly attached or directly immobilized on the support using standard methods (see, for example, Harlow and Lane, Antibodies, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1988); Biancala et al., Eettera in Peptide Science 7(291): 297(2000); MacBeafh et al., Science 289: 1760-1763 (2000); Cass et al., ed., Proceedings of the Thirteenth American Peptide Symposium; Leiden, Escom, 975-979 (1994); U.S.
• the ligand(s) are synthesized on the surface of the support, which is advantageous in generating peptide libraries.
• the ligand(s) can be chemically conjugated to the support or can be attached via linkers, beta alanine, glycine, polymers containing glycine-serine, short chain hydrocarbons of the formula -(CH 2 )-, polyethylene glycol, epsilon amino caproic acid, and linkers comprising -O(CH )n, wherein n is 1-30.
• the ligand(s) can be attached by one or by several different cleavable linkers, e.g., photolabile or acid labile moieties, enabling the selective detachment of a population of ligands for analysis.
• Ligands can be used, for example, as affinity purification media for proteins and enantiomeric separation (e.g., to concentrate, isolate, detect, characterize, quantify, or identify entities in a sample), as diagnostic therapeutic tools, catalysts and enhancers of chemical reactions, and as selective stabilizers of proteins.
• the ligand-support complexes are contacted with a mixture comprising a plurality of entities under conditions that allow at least one entity to bind to at least one ligand-support complex, thereby forming at least one entity-ligand-support complex.
• conditions as recognized by one of ordinary skill in the art, depend upon the mixture (entities) and the ligands themselves, in addition to other factors, such as pH, temperature, contact time, salt concentration, and the like. Determination of suitable conditions that allow at least one entity to bind to at least one ligand-support complex is within the ordinary skill.
• multiple entity-ligand support complexes are formed.
• At least one entity-ligand-support complex is formed, at least one entity-ligand-support complex and the ligand-support complexes are separated from the unbound entities.
• unbound entities refers to the entities of the mixture that do not bind or are loosely bound to any of the ligand-support complexes.
• Means of separating at least one entity-ligand-support complex from the unbound entities are known in the art and, include, for instance, centrifugation, serial dilution, filtration, dialysis, and washing in a chromatographic format.
• the inventive methods can optionally comprise a sub-pooling step, wherein at least one entity-ligand-support complex and the ligand-support complexes are separated into several pools or sub-populations.
• the sub-pooling step achieves on average about 10-500 entity-ligand-support complexes and ligand-support complexes per pool. More preferably, the average number of complexes per pool is 20-100. Most desirably, the complexes are sub-pooled, such that 50 complexes are present in each pool.
• inventive methods can optionally comprise, after the sub- pooling step, an eluting step, wherein the entities of the multiple entity-ligand-support complexes are dissociated from the complexes, and the ligand-support complexes are subsequently removed from the pools.
• an eluting step wherein the entities of the multiple entity-ligand-support complexes are dissociated from the complexes, and the ligand-support complexes are subsequently removed from the pools.
• the invention provides a second method of screening a mixture for active entities.
• This method comprises (i) providing a plurality of ligands, wherein each ligand is attached to a support to form a plurality of ligand-support complexes, (ii) contacting the ligand-support complexes with a mixture comprising a plurality of entities under conditions that allow at least one entity to bind to at least one ligand-support complex, thereby forming at least one entity-ligand-support complex, (iii) separating at least one entity-ligand-support complex and the ligand-support complexes from the unbound entities, (iv) separating at least one entity-ligand-support complex and the ligand-support complexes into pools, (v) dissociating at least one entity from at least one separated entity- ligand-support complex, (vi) removing from the pools the ligand-support complexes or the at least one dissociated entity of step (v), (vii) assaying at least one dissociated entity of step (v) for an activity, (viii) detecting
• One of the advantages of this inventive method is that the entity being tested for activity dissociates from the ligand-support complex and is, thus, in an unbound state. It is therefore, uninhibited by the ligand and/or support and is free to interact with and act on the enzyme substrate or cells used in the activity assay.
• the degree of dissociation of the entity from the ligand-support complex need not be 100% or a complete dissociation, as it is possible that an activity of the entity can be detected with only some of the entity dissociated from the ligand-support complex.
• the method could optionally further comprise, after the sub- pooling step, a step, wherein a semi-solid or viscous material is added to each pool, wherein the entity of at least one entity-ligand-support complex dissociates from the complex and diffuses into the material, thereby forming a concentration gradient of the entity, wherein the concentration of the entity gradually decreases as the distance from the ligand-support complex from which the entity dissociates increases.
• Suitable semi-solid materials for use in this method include, for instance, agarose, gelatin, glycerol, polyethylene glycol, acrylamide, and fibrin sealant.
• the semi-solid material is 0.5% w/v agarose.
• the invention provides a third method of screening a mixture for active entities.
• This method comprises (i) providing a plurality of ligands, wherein each ligand is attached to a support to form a plurality of ligand-support complexes, (ii) contacting the ligand-support complexes with a mixture comprising a plurality of entities under conditions that allow at least one entity to bind to at least one ligand-support complex, thereby forming at least one entity-ligand-support complex, (iii) separating at least one entity-ligand-support complex and the ligand-support complexes from the unbound entities, (iv) separating at least one entity-ligand-support complex and the ligand-support complexes into pools, (v) adding a semi-solid or viscous material to each pool, wherein the entity of the at least one separated entity-ligand-support complex dissociates from the complex and diffuses into the material, thereby forming a concentration gradient of the entity,
• the advantages offered by this method also include the entity being in a free, unbound state, uninhibited by the ligand-support complex to interact with or act on an enzyme substrate or cells used in the activity assay.
• this method offers the advantage of being able to test the activity of the entity in a dose-dependent manner.
• the mixture is screened for active entities by assaying at least one entity-ligand-support complex for an activity.
• active entity refers to any entity of the mixture that exhibits the assayed activity or a serendipitous result. In this regard, most entities of a given mixture have the potential of being an active entity, depending upon the particular activity being assayed in the method.
• the active entity will also be active in the sense that the entity binds to a ligand- support complex.
• the assayed activity can be any biological, physical, chemical, or biochemical activity, provided that the activity results in a detectable signal, such as the enzymatic modification of a substrate.
• a chemical activity e.g., an activity directly related to the chemical composition of the entity, can be employed.
• the entity can be identified by the presence of specific chemical subunits or moieties or chemical structures.
• Physical properties useful in detection methods include, for example, spectral signal, which can be determined via fluorescence or mass-spectrometry, respectively.
• the means of detection need not detect the activity of the entity alone, but can selectively identify activity or activities of more than one entity or of an entity complex, e.g., an entity complexed with other biological entities such as co-factors or enzymes.
• the activity can be an enzyme activity or inhibition of an enzyme activity.
• the inventive method can comprise performing an enzyme activity assay to characterize an entity on the basis of biological activity. An enzyme substrate is applied to at least one entity-ligand-support complex under conditions which allow for enzymatic modification of the substrate by the entity to form a product. The product is then detected, thereby identifying the presence of the entity. Alternatively, the lack of product formation could be detected in order to identify active entities that inhibit a given enzyme activity.
• Enzyme activity assays are further described in, for example, Haugland, supra.
• the activity alternatively can be an effect on a cell, a cell population, a tissue, or a whole organism.
• a cell-based assay is performed wherein at least one entity-ligand-support complex is contacted with cells on which the entity exerts some observable biologic effect.
• the preferred entity can be an antibacterial agent.
• the ligand of the inventive methods binds potential active sites of the antibacterial agent, thereby separating the antibacterial entity from a mixture (e.g., a library of potential therapeutics). A lawn of bacteria is applied to the entity, and the antibacterial entity is detected by zones of bactericidal activity.
• the effect can be, for instance, cell migration, cell proliferation, cell death, cell differentiation, cell cycle entry, cell cycle arrest, apoptosis, cell lysis, growth arrest, cell survival, a change in an intracellular signaling pathway, antigen expression, gene upregulation, gene downregulation, or a phenotypic change in response to an agent.
• the cell, cell population, tissue, or whole organism is diseased or is derived from a diseased source.
• the diseased cell population, tissue, or whole organism can be diseased with any disease, malady, infection, and the like.
• the disease is cancer, diabetes, an autoimmune disease, osteoporosis, or lung disease.
• the diseased cell population, tissue, or whole organism is infected with a parasite, virus, or bacteria.
• the diseased cell population, tissue, or whole organism can also be wounded, burned, scarred, or in a state of healing.
• the diseased cell also can be a protozoan, a nematode, T. cruzi, and leishmania.
• the precise techniques for detecting the assayed activity will depend on the activity itself. For instance, if the activity is cell growth, then detection of the activity may simply comprise a cell count using a hemocytometer, visual inspection, or radioactive isotope uptake. If the activity is the production of a color-tagged product, then detection may involve detection of the color via ultraviolet- visible (UV-VIS) spectroscopy. It is well within the ordinary skill for one to determine suitable techniques for detecting the assayed activity.
• UV-VIS ultraviolet- visible
• At least one entity-ligand-support complex having the entity, which exhibited the detected activity is selected.
• the term "selecting" and words stemming therefrom as used herein refers to the identification of the entity-ligand-support complex, or the pool within which it resides. In the latter case, the assay may be repeated with just the constituents of the selected pool until a single entity-ligand-support complex is selected.
• An advantage of the inventive methods is the ability to identify and/or characterize active entities on the basis of biological, physical, biochemical, or chemical activity, without prior knowledge of the entity's molecular identity. Accordingly, the entity can display a biological activity and need not undergo processing (e.g., heat-inactivation) prior to practicing the inventive methods. Also, there is no need to remove more abundant proteins like albumin or active entities like immunoglobulins. Likewise, the ability of an entity to affect more specific cellular functions (e.g., production of particular proteins or other cellular constituents) might be enhanced or diminished in the assay medium, thereby providing valuable characteristics of the entity. Furthermore, the entity might be a cell that proves resistant to cytotoxic agents. Thus, the invention provides methods for the identification of novel active entities or unknown active entities (i.e., proteins not identified prior to practicing the inventive method) with specific biological activities. Also, the invention provides identification of novel or unknown biological activities for known proteins.
• the way in which the methods can achieve identification of the active entities can involve determining the chemical identity of the ligand(s) that bind(s) to the entity exhibiting the assayed activity. Suitable methods of determining the chemical identity of the ligand(s) are known in the art and include, for example, mass spectrometry, Edman degradation sequencing, and the like. Once ligands have been identified as having specificity for particular entities, those ligands can be resynthesized and used to capture, isolate, detect, and/or characterize entities using, for example, chromatographic separation.
• the inventive methods further comprise providing multiple copies of the identified ligand and attaching each copy of the identified ligand to a support, thereby obtaining multiple ligand-support complexes.
• the multiple-ligand support complexes are allowed to contact a composition containing multiple copies of the entity under conditions that allow the ligand-support complexes to bind to multiple copies of the entity, thereby forming multiple entity-ligand-support complexes.
• the entities are dissociated from the entity-ligand-support complexes and, if desired, subjected to additional rounds of screening and/or characterization.
• the composition containing multiple copies of the entity used to isolate and purify the entity can be the same as the mixture that was originally used to identify the entity as an active entity. For instance, if plasma was the mixture that was originally contacted with the ligand-support complexes, then the same source of plasma can be used to isolate and purify the entities in subsequent steps.
• the method can further comprise the step of determining the chemical or physical identity of the entity or at least further characterizing the entity, such as by performing mass spectrometry of the entity.
• characterization and words related thereto as used herein refer to the identification of any distinctive quality or trait of a entity, and do not require that the precise chemical identity, e.g., the molecular formula, chemical structure, conformation, nucleotide sequence or amino acid sequence, of the entity is elucidated.
• the identified ligands also can be. used in diagnostic assays, to immobilize or selectively transfer entities, and as pseudo- or synthetic receptors (see, e.g., Still, Ace. Chem. Res., 29: 155-163 (1996)). Additionally, the ligands themselves can be used as therapeutic agents, catalysts, and the like.
• DEPFMU 6,8-difluoro-4-methylumbelliferyl
• ICAT isotope coded affinity tag
• UN- VIS ultraviolet-visible
• SAP streptavidin-alkaline phosphatase
• FITC fluorescent isothiocyanate
• ATCC American Type Culture Collection
• JL-2 interleukin-2
• P ⁇ PP p- nitro phenyl phosphate
• PPV porcine parvovfrus
• PVDF polyvinylidene fluoride
• AP alkaline phosphatase
• PI propidium iodide.
• This example demonstrates the use of the inventive methods to screen cytokine- spiked plasma for factors that support cell growth.
• the cells used in this assay were NK-92 cells (obtained from ATCC, Manassas, VA).
• the NK-92 cell line is a human cytotoxic T-cell line, which requires exogenous Interleukin-2 (IL-2) in order to grow.
• IL-2 Interleukin-2
• Normal cell culture medium containing serum or healthy human plasma does not have IL-2 to support NK-92 growth. Therefore, NK-92 cells normally are maintained in culture in medium supplemented with 5 ng/ml recombinant mouse EL-2.
• rat anti-human IL-2 antibody As a model for beads bearing an EL-2 specific ligand, 300 ⁇ g of rat anti-human IL-2 antibody (Pharmingen, San Diego, CA) was cross-linked onto 400 ⁇ l of Protein G sepharose beads (Pierce, Rockford, EL). The beads were incubated overnight with a mixture of plasma into which a natural, secreted cytokine mixture was spiked. This mixture contained several cytokines and was derived from human monocytes and leukocytes that had been induced to secrete cytokines with phytohemagglutanin and ciprofloxacin. Media from these induced cells was subsequently pooled. The concentration of IL-2 in the spiked plasma was approximately 4 ng/ml.
• the beads were washed with ⁇ 50 column volumes of PBS + 0.1% Tween-20 (Sigma Aldrich, St. Louis, MO). Beads that were incubated with 4 ng/ml pure recombinant human EL-2 were also included. Controls were beads bearing the antibody that were not incubated with EL-2 as a negative control and cultures to which soluble IL-2 was added as a positive control.
• NK-92 cells were plated at 1 x 10 5 cells per well in 24- well plates and allowed to grow in media without added IL-2 for 24 hours to deplete the intrinsic EL-2 stores. Approximately 100 beads were added to each well. The cultures were allowed to grow for 96 hours. Cells were collected from each culture and the cell number determined by counting live cells in a hemocytometer, excluding dead cells by trypan blue exclusion. Wells in which IL-2 was provided to the cells from ligands, either as recombinant protein or purified from the secreted mixture, showed an 8- to 12-fold increase in cell number compared with the negative control.
• cytokine mixtures have been screened for proteins that support the survival of NK-92 cells using a library of hexamer peptide ligands.
• a natural, secreted cytokine mixture derived from isolated lymphocytes and monocytes (ImmunoRx, Inc, Farmington, NY) was used as a starting material. This mixture contained many cytokines that are released in response to biological induction and are not present in normal sera or culture media.
• the endpoints of this assay are both biological and fluorescent as described in Example 2.
• a large clump of cells grew in close association with a bead.
• PI propidium iodide
• One of these beads (and a few others from similar wells that supported growth) was collected and the presence of EL-2 on the beads was confirmed by modified antibody detection in a "bead blot" assay. Briefly, the beads are arrayed in agarose, the proteins transferred off the beads onto a PVDF membrane by capillary transfer in elution buffer, and the membrane probed with anti-IL-2 antibodies to detect EL-2.
• Eleven beads, including three potential positives were recovered, cleaned, re-incubated with the identical starting material and cultured individually with the same cell line. Two of three potential positive beads reconfirmed their activity in the deconvolution assay. An additional bead was sequenced and the associated ligand identified as the sequence GVASED (SEQ ED NO: 9). A resin with this ligand was synthesized and found to bind EL-2. The complete starting material will be fractionated on the resin and the bound proteins will be analyzed to identify additional proteins that may be purified on the resin and which contribute to the activity.
• IL-2 is known to be present in the starting material there are also numerous other cytokines present at various levels on the beads and in the assay that may be contributing to cell survival.
• NK-92 cells have been tested for their lack of responsiveness to IL-1 and several other known cytokines; however, they may be responsive to other, as yet undiscovered cytokines that are present in the starting material.
• screening for factors that are protective against cytotoxic agents and poisons can clearly be accomplished in this type of assay in which cell growth and PI exclusion are endpoints.
• This example demonstrates the use of the inventive methods to screen spiked plasma for alkaline phophatase enzyme activity.
• Streptavidin-alkaline phosphatase 500 ng was spiked into 1 ml of pooled human plasma. The spiked plasma was incubated with ToyoPearl AF-
• Amino-650M beads (Tosoh BioSciences, Montgomeryville, PA) either with or without the ligand HPQFLS (SEQ ID NO: 1) (synthesized at Peptides International, Louisville, KY), a sequence known to bind streptavidin.
• the beads were allowed to incubate with spiked or unspiked plasma for 1 hr at room temperature, after which they were washed with HEPES buffer with 0.1% Tween-20, pH 7.2.
• Ligand-bearing beads were also incubated with saline alone as a control.
• the beads were distributed into wells of a 384- ell plate with 6 HPQFLS beads per well and 120 ⁇ l of HEPES buffer containing 250 mM NaCl and 0.05% Tween-20 buffer. The beads were allowed to incubate for 30 hours at room temperature. [0063] The liquid was removed from the wells and tested for the presence of alkaline phosphatase activity with p-nitro phenyl phosphate (PNPP) (Sigma- Aldrich), a colored phosphatase substrate. Yellow color developed only in those wells with buffer from ligand beads that had been exposed to AP-spiked plasma; no color was observed in the control wells.
• PNPP p-nitro phenyl phosphate
• a FITC-labeled streptavidin (Pierce) was also incubated with the HPQFLS (SEQ ID NO: 1) beads as described above. The beads were then incubated with 120 ⁇ l of 20 mM citrate 140 mM NaCl buffer for 21 hours. The buffer was collected, and fluorescence was measured in a fluorometer. The increase over baseline was 11.6%. After an additional 3 days of elution, the fluorescence increased 22.7% over baseline.
• the cell line used in this assay was the human, malignant melanoma cell line SK-MEL 28.
• the combinatorial library was synthesized on ToyoPearl 650-M epoxy resin from Tosoh BioScience. This library was designed to have the ligands linked to the resin through the sulfhydryl group of a cysteine derivative.
• the starting material used as a source of IgG, IgA and IgM antibodies was a plasma fraction I+ ⁇ +m paste that had been further fractionated to remove fibrinogen.
• the beads were incubated with paste and washed, and between 50 -150 beads were incubated with 40 ⁇ l SK-MEL28 cells that had been plated at 1 x 10 4 cells/ml in wells of a 384- well plate.
• the media contained propidium iodide (PI), a red-fluorescent dye that is taken up only by dead cells. Images of each well were taken at 1 hour intervals for 160 hours with single cell resolution. The images were analyzed for uptalce of PI over the time period, and wells in which a cumulative increase in PI uptake that was 2 standard deviations above the mean of the population were selected as being of particular interest.
• PI propidium iodide
• the beads in these wells were harvested, cleaned, re-loaded with a fresh aliquot of the original starting material, and incubated with 1-2 beads per well with the same concentration of cells and imaged in the same way as the original assay. Beads in wells in which an increase in death above 2 standard deviations above the mean of the population was seen were collected and the associated ligand sequenced.
• This example demonstrates the use of the inventive methods to screen a mixture of antibodies for antibodies of an immunized mammal.
• Human plasma-derived intravenous immunoglobulin (IgG) was manufactured from pools of up to 60,000 donors and included vast varieties of antibodies with diverse affinities, some of which bind to cell surface receptors. Populations of antibodies can also be raised that are directed against cell surface receptors by immunizing mice with membrane preparations. The antibodies can be used as screening starting materials to identify their receptor epitopes in methods such as the "bead blot" receptor-binding antibodies may be useful for the upregulation of PON1. Specific epitopes for antibodies in a differential screen were identified using purified IgG preparations from normal and immunized mice. One cohort of mice was immunized with ovalbumin; unimmunized mice were used as controls.
• IgG preparations from each group were purified from pooled sera using affinity chromatography on protein G sepharose (Pierce), and the two populations were differentially labeled with either Alexa 488 (green) or Alexa 568 (red) dye (Molecular Probes, Eugene OR).
• the two IgG populations were then mixed and incubated with a library of combinatorial hexamer ligands.
• the beads were washed extensively and observed under a fluorescence microscope. The majority of beads that fluoresced had both red and green signals, indicating that they bound antibodies that were present in both populations. Beads that fluoresced only red were indicative of antibodies that were present only in the immunized population. These were collected and sequenced.
• ILRVIR (SEQ ID NO: 2) has homology with the ovalbumin sequence RTINKWRF (SEQ ID NO: 3) , IFDKVQG (SEQ ID NO: 4) homology with RFDKLPGFG (SEQ ID NO: 5) and PPFRIHG (SEQ ED NO: 6) homology with MPFRVITE (SEQ ED NO: 7).
• RTINKWRF SEQ ID NO: 3
• IFDKVQG SEQ ID NO: 4
• RFDKLPGFG SEQ ID NO: 5
• PPFRIHG SEQ ED NO: 6
• a ligand that binds HDL was screened for its ability to purify paraoxonase from plasma by measuring the enzyme's activity. Paraoxonase is implicated in protecting against the build up of atherosclerotic plaque, as well as detoxification of some nerve poisons (sarin) and insecticides. 5 ⁇ g of the ligand 2'-naphthyl-alanineWLHAN (SEQ ED NO: 8) was incubated with 100 ⁇ l of 1:10 diluted rabbit serum in PBS for one hour at 37° C. The beads were centrifuged to remove the supernatant and the supernatant stored in eppendorf tubes.
• the resin bead pellets were resuspended in an equal volume of paraoxonase assay buffer.
• Paraoxonase activity of the supernatants was measured using a sensitive, specific, fluorescent substrate.
• 100 ⁇ M DEPFMU (6, 8-difluoro-4-methylumbelliferyl) was mixed with 10 ⁇ l supernatant, 10 ⁇ l of bead suspension, or 10 ⁇ l serum in a standard microtiter plate for 20 minutes at 37° C.
• Hydrolysis of DEPFMU was quantified by measuring fluoresce at 355 rrm emission and 460 nm excitation using a commercial fluorometer. Hydrolysis was quantified compared with a standard curve of DEPFMU activity.
• This example demonstrates the use of the inventive methods to screen libraries for ligands that bind virus based on screening for virus-associated cytotoxicity.
• Ligands that bind porcine parvovirus (PPV) were identified using the inventive methods. This assay was a modification of the classical plaque assay, in which infectivity is measured by viral plaques formed by lysis of susceptible mammalian cells in culture. As a model for ligands that bind virus, 2 ⁇ l of polyclonal porcine anti-PPV antibody was conjugated to Protein A sepharose beads according to the manufacturer's protocol (Pierce). The beads were incubated with 8 logs of PPV purified from PK13 cells by serial centrifugation.

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